Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture.METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined.RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability.CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines.     

Duodenal phytohaemagglutinin (red kidney bean lectin) stimulates gallbladder contraction in humans

Aim: Lectins, carbohydrate‐specific proteins without enzymatic activity on the ligand, are daily ingested plant proteins which survive the passage through the gastrointestinal tract in a biologically active form. Their binding to glycan determinants of natural glycoconjugates can trigger biological effects. The lectin phytohaemagglutinin (PHA) is abundantly present in red kidney beans and induces cholecystokinin (CCK) release in rats. The aim of the study was to investigate the effect of intraduodenal administration of PHA on plasma CCK levels and gallbladder contraction in humans and to elucidate potential mechanisms of action. Methods: Five healthy volunteers underwent four studies. After a basal intraduodenal saline infusion for 30 min, PHA or heat‐inactivated PHA was infused in increasing doses: 150 μg, 1.5 mg and 15 mg for 30 min each. Intravenous saline, CCK₁ receptor antagonist dexloxiglumide or atropine were administered in random order. Gallbladder volumes were measured by ultrasonography and plasma CCK levels by radioimmunoassay. Results: Intraduodenal PHA induced gallbladder contraction in a dose‐dependent fashion starting with the lowest dose. The highest dose reduced the gallbladder volume to 65.3 ± 9.4% of basal volume (P < 0.001) whereas heat‐inactivated PHA did not have any effect. Blocking CCK₁ or muscarinic receptors completely abolished PHA‐stimulated gallbladder contraction (dexloxiglumide 208.7 ± 23.7%; atropine 104 ± 7.0% of basal volume) while none of the treatments affected CCK levels. Conclusion: Duodenal administration of PHA potently stimulates gallbladder contraction in humans. This contraction is mediated via cholinergic pathway.     

Increased expression and altered intracellular distribution of adhesion/growth-regulatory lectins galectins-1 and -7 during tumour progression in hypopharyngeal and laryngeal squamous cell carcinomas

Aims:  To examine the level of expression of the pleiotropic regulators galectins-1 and -7 in relation to neoplastic progression of hypopharyngeal (HSCCs) and laryngeal (LSCC s ) squamous cell carcinomas.
Methods and results:  The presence of galectins-1 and -7 was investigated using quantitative immunohistochemistry in (i) a series of 78 HSCCs by comparison with 17 normal epithelia (N_E), 26 low-grade dysplasia (low_D) and 27 high-grade dysplasia (high_D) and (ii) a series of 56 LSCCs by comparison with 50 N_E, 23 low_D and 29 high_D. Galectin-1 positivity expressed as a percentage of cells was significantly higher in carcinomas (HSCCs and LSCCs) than in N_E, low _ D or high _ D ( P  < 10⁻⁶). Galectin-7 expression was elevated in low _ D ( P  = 0.0004) compared with N_E and in carcinomas (HSCC) compared with high _ D ( P  = 0.0002). Tumour progression from high_D to carcinomas was associated with a shift of galectin-1 localization from the nucleus towards the cytoplasm. Increased expression of galectin-7 in dysplasias was accompanied by a shift from the cytoplasmic compartment (N_E) to the nucleus (low_D and high_D).
Conclusions:  Our data reveal an association between the level of presence of galectins-1 and -7 and neoplastic progression of HSCCs and LSCCs. Moreover, inverse shifts between nuclear and cytoplasmic positivity intimating functional divergence were detected.     

Galectin-3 upregulation during tumor progression in head and neck cancer

Objectives/Hypothesis: To examine the level of expression of galectin-3 in relation to neoplastic progression of hypopharyngeal squamous cell carcinomas (HSCCs) and laryngeal squamous cell carcinomas (LSCCs). Study Design: Retrospective study. Methods: Using a polyclonal antibody against galectin-3 without cross-reactivity to other galectins, we analyzed the presence of galectin-3 using quantitative immunohistochemistry in i) a series of 79 HSCCs compared with 16 normal epithelia, 20 low-grade dysplasia (Low_D) and 25 high-grade dysplasia (High_D) and in ii) a series of 58 LSCCs compared with 34 normal epithelia, 12 Low_D, and 18 High_D. In parallel, galectin-3 expression was studied using Western blotting on a series of 19 fresh biopsies from patients presenting a head and neck tumor. Results: Western blotting excluded a notable degree of proteolytic truncation of galectin-3 in situ. Immunohistochemical galectin-3 positivity expressed as percentage of cells was significantly higher in LSCCs and HSCCs than in Low_D (P = .01) or High_D (P = .0002), respectively. Increased expression of galectin-3 in HSCCs was accompanied by a shift from the cytoplasmic compartment to the nucleus (P = .007). In intertumor-type comparison, laryngeal carcinomas presented nuclear presence of galectin-3 only rarely (1 of 58 cases in laryngeal cancer vs. 27 of 79 cases in hypopharyngeal cancer, P = .00006) and a comparatively low labeling index (P < 10⁻⁶). Conclusions: Our data reveal an association between level of presence of galectin-3 and neoplastic progression of HSCCs and LSCCs.     

Galectin 7 (p53-Induced Gene 1): A New Prognostic Predictor of Recurrence and Survival in Stage IV Hypopharyngeal Cancer

Background Eighty percent of hypopharyngeal squamous cell carcinoma patients have advanced stages (III and IV) of the disease, and biological markers are required to predict high-risk head and neck squamous cell carcinoma patients in need of highly aggressive treatments after surgery to improve the survival rate. We analyzed the potential prognostic value of galectin 7 in a series of 81 stage IV hypopharyngeal SCCs because galectin 7 is an emerging marker involved in the epidermal development of pluristratified epithelia and in epidermal cell migration. Methods The immunohistochemical expression of galectin 7 was determined on a series of 81 stage IV hypopharyngeal SCCs and was compared with that of galectins 1 and 3. Results High levels of galectin 7 expression were associated with rapid recurrence rates and dismal prognoses in these 81 stage IV hypopharyngeal SCCs, a feature not observed with galectin 3 and one observed weakly, if at all, with galectin 1. Conclusions These data suggest that the immunohistochemical determination of galectin 7 expression in the case of high-risk hypopharyngeal cancers is a meaningful tool to identify patients who should benefit from aggressive postsurgical adjuvant therapy after surgery, including not only radiotherapy, but also chemotherapy.     

Combined cimetidine and temozolomide, compared with temozolomide alone: significant increases in survival in nude mice bearing U373 human glioblastoma multiforme orthotopic xenografts

OBJECT: Malignant gliomas consist of both heterogeneous proliferating and migrating cell subpopulations, with migrating glioma cells exhibiting less sensitivity to antiproliferative or proapoptotic drugs than proliferative cells. Therefore, the authors combined cimetidine, an antiinflammatory agent already proven to act against migrating epithelial cancer cells, with temozolomide to determine whether the combination induces antitumor activities in experimental orthotopic human gliomas compared with the effects of temozolomide alone. METHODS: Cimetidine added to temozolomide compared with temozolomide alone induced survival benefits in nude mice with U373 human glioblastoma multiforme (GBM) cells orthotopically xenografted in the brain. Computer-assisted phase-contrast microscopy analyses of 9L rat and U373 human GBM cells showed that cimetidine significantly decreased the migration levels of these tumor cells in vitro at concentrations at which tumor growth levels were not modified (as revealed on monotetrazolium colorimetric assay). Computer-assisted microscope analyses of neoglycoconjugate-based glycohistochemical staining profiles of 9L gliosarcomas grown in vivo revealed that cimetidine significantly decreased expression levels of endogenous receptors for fucose and, to a lesser extent, for N-acetyl-lactosamine moieties. Endogenous receptors of this specificity are known to play important roles in adhesion and migration processes of brain tumor cells. CONCLUSIONS: Cimetidine, acting as an antiadhesive and therefore an antimigratory agent for glioma cells, could be added in complement to the cytotoxic temozolomide compound to combat both migrating and proliferating cells in GBM.